Millions of people suffer from xerostomia, or dry mouth resulting from lack of saliva, causing a decreased quality of life resulting from multiple symptoms, including increased dental caries, oropharyngeal infections, difficulties with swallowing (dysphagia) and digestion (mucositis), loss of taste, and pain. As current treatments for these problems are inadequate (1), there is considerable interest in creating an artificial salivary gland. Maintenance of salivary acinar cell differentiation and function in vitro is criticl to the successful engineering of such constructs; however, this breakthrough has not yet been achieved. This is primarily due to the current lack of basic scientific knowledge regarding the specific extracellular signals that are required to maintain or induce acinar cell differentiation, which remains a substantial limitation in the ability to engineer a functional artificial salivary gland. An in vitro assay system is needed to identify required extracellular signals. We hypothesize that the combination of chemically-modified nanofibers presented to cells via microscale patterning in a 3D scaffold will support salivary acinar cell function. This application proposes an innovative, interdisciplinary strategy to create a high-throughput, non-invasive assay system that will be used to sense salivary acinar cell secretory function in live cells grown on scaffold materials, which has not been previously possible. A MEMS-based saliva sensor will be produced to accurately measure the concentration of a salivary secretory protein secreted by cells grown on unique scaffold combinations. A novel multiplexed immunocytochemistry method will be used to assess the extent of differentiation in fixed cells following the biosensor assay. Using an innovative combination of methods, nanotopography, micropatterning, and chemical signaling will be independently modulated, such that specific combinations of parameters will be identified that support acinar cell function. Scaffolds will be assessed for their ability to maintin acinar differentiation or promote re-differentiation using a combination of primary cells and cell lines. We will address this hypothesis in four specific aims: Aim 1: Develop a high-throughput MEMS probe array assay system to evaluate acinar cell function in vitro. Aim 2: Identify the ideal nanofiber configuration to support salivary acinar cell differentiation. Aim 3: Optimize nanofiber surface properties to enhance salivary gland acinar cell function. Aim 4: Identify an optimal microtopography to promote acinar cell function. The in vitro salivary gland construct produced in this application will be useful to identify pathways regulating acinar function and can be applied for drug screening. The principles developed as a result of this application will be applied in the future towards engineering of artificial glands designed to replicate salivary gland and other complex branching organs. PUBLIC HEALTH RELEVANCE: In this application, optimized micropatterned scaffolds having a nanofiber-coated surface will be engineered to develop a smart scaffold for functional saliva-secreting salivary gland acinar cells. The methods developed here will be useful for application in high-throughput drug screening. This work will be applied in the future to develop an orally implantable device to enhance salivary flow in patients suffering from salivary hypofunction.